Insulin-like growth factor-1 (IGF-1) is a polypeptide of 70 amino acids with a molecular weight of 7648 daltons. This single chain protein has three intrachain disulfide bridges. These disulfide bonds, along with numerous hydrogen bonds and hydrophilic interactions, maintain the compact tertiary structure of this molecule. However, Meng et al. [see J. Chrom., 443: 183 (1988)] have shown that, upon reduction and reoxidation, IGF-1 can refold in a variety of ways, forming as many as 15 monomeric configurations. Consequently, attempts to produce this peptide by recombinant means in E. coli host expression systems provides a complicated mixture of product forms which must be purified for further use [see Grossgian and Friers in Gene, 18: 199 (1985)].
Insulin-like growth factor-1 belongs to a heterogeneous family of peptides which share some of the biological and chemical properties of insulin, but which are antigenically distinct from insulin. Currently available experimental evidence suggests that IGF-1 promotes growth by mediating the effects of growth hormone. Thus, such processes as skeletal growth, cell replication and other growth related processes are affected by IGF-1 levels.
Physiological concentrations of IGF-1 have been shown to be influenced by such conditions as thyroid disease, diabetes and malnutrition [see Preece, in Horm. Blood, 4: 108 (1983)]. IGF-1 has also been shown to act synergistically with other growth factors, for example, in accelerating the healing of soft and mesenchymal tissue wounds [see Lynch et al., in J. Clin. Periodontol., 16: 545 (1989) and Lynch et al., in Proc. Natl. Acad. Sci. USA, 84: 7696 (1987)], and in enhancing the growth of mammalian cells in serum-free tissue culture medium [see Burleigh and Meng, in American Biotech. Lab., 4: 48 (1986)].
Considering the many clinical and research applications of IGF-1, a ready supply of IGF-1, such as that which would result from fermentation of IGF-1 expressing recombinant organisms, will be of great value to the medical and biotechnology fields.
Since isolation from natural sources is technically difficult, expensive, and time consuming, recent efforts have centered on the development of efficient recombinant methods for the production of IGF-1.
Of the hosts widely used for the production of heterologous proteins, probably E. coli and Saccharomyces cerevisiae (Baker's yeast) are the best understood. However, E. coli does not possess the ability to produce disulfide bonds in proteins, thus proteins such as IGF-1 frequently are not stable in the presence of endogenous bacterial proteases, and tend to aggregate into inactive complexes. As a result of this inability to produce disulfide bonds, IGF-1 produced in E. coli has to be extracted and then the disulfide bonds have to be formed by oxidation. This results in a complicated mixture of 15 different forms of IGF-1, which must be separated. Consequently, the yield of purified product is very low (Grossgian and Friers, supra). Furthermore, in order to produce in E. coli IGF-1 molecules which contain the authentic N-terminal amino acid, i.e., glycine, and not the initiating methionine present on the primary translation product, it is necessary to express IGF-1 in E. coli as a fusion protein. Cleavage of mature IGF-1 from the initially produced fusion protein necessitates an additional step in the production process.
Yeasts can offer clear advantages over bacteria in the production of heterologous proteins, which include their ability to secrete heterologous proteins into the culture medium. Secretion of proteins from cells is generally superior to production of proteins in the cytoplasm. Secreted products are obtained in a higher degree of initial purity; and further purification of the secreted products is made easier by the absence of cellular debris. In the case of sulfhydryl-rich proteins, there is another compelling reason for the development of eukaryotic hosts capable of secreting such proteins into the culture medium: their correct tertiary structure is produced and maintained via disulfide bonds. This is because the secretory pathway of the cell and the extracellular medium are oxidizing environments which can support disulfide bond formation [Smith, et al., Science, 229: 1219 (1985)]; whereas, in contrast, the cytoplasm is a reducing environment in which disulfide bonds cannot form. Upon cell breakage, too rapid formation of disulfide linkages can result in random disulfide bond formation. Consequently, production of sulfhydryl-rich proteins, such as IGF-1, containing appropriately formed disulfide bonds, can potentially be best achieved by transit through the secretory pathway.
Gellerfors et al., in J. Biol. Chem., 264: 11444-11449 (1989), describe the production of IGF-1 in S. cerevisiae under the control of the S. cerevisiae actin promoter. The IGF-1 product is encoded by autonomously replicating plasmid-borne DNA. In a similar study, Bayne et al., in Gene, 66: 235-244 (1988), describe the production of IGF-1 in S. cerevisiae under the control of the S. cerevisiae alpha mating factor promoter. The latter authors report yields of IGF-1 which are quite low, with production of only about 2 mg of IGF-1 per liter of fermentation broth being reported.
In view of the problems usually encountered with up-scaling the production of heterologous proteins in autonomous plasmid-based yeast systems, such as S. cerevisiae, and the difficulties observed in reported work in S. cerevisiae, no motivation is provided by the art for one to further pursue the production of IGF-1 in S. cerevisiae.
To overcome the major problems associated with the expression of recombinant gene products in S. cerevisiae (e.g., loss of selection for plasmid maintenance and problems concerning plasmid distribution, copy number and stability in fermentors operated at high cell density), a yeast expression system based on methylotrophic yeast, such as for example, Pichia pastoris, has been developed. A key feature of this unique system lies with the promoter employed to drive heterologous gene expression. This promoter, which is derived from a methanol-responsive gene of a methylotrophic yeast, is frequently highly expressed and tightly regulated (see, e.g., European Patent Application No. 85113737.2, published Jun. 4, 1986, under No. 0 183 071, now issued in the United States as U.S. Pat. No. 4,855,231). Another key feature of expression systems based on methylotrophic yeast is the ability of expression cassettes to stably integrate into the genome of the methylotrophic yeast host, thus significantly decreasing the chance of vector loss.
Although the methylotrophic yeast, P. pastoris, has been used successfully for the production of various heterologous proteins, e.g., hepatitis B surface antigen [Cregg et al., Bio/Technology 5, 479 (1987)], lysozyme and invertase [Digan et al., Developments in Industrial Microbiology 29: 59 (1988); Tschopp et al., Bio/Technology 5: 1305 (1987)], endeavors to produce other heterologous gene products in Pichia, especially by secretion, have given mixed results. At the present level of understanding of methylotrophic yeast expression systems, it is unpredictable whether a given gene can be expressed to an appreciable level in such yeast or whether the yeast host will tolerate the presence of the recombinant gene product in its cells. Further, it is especially difficult to foresee if a particular protein will be secreted by the methylotrophic yeast host, and if it is, at what efficiency. Even for the non-methylotrophic yeast, S. cerevisiae, which has been considerably more extensively studied than P. pastoris, the mechanism of protein secretion is not well defined and understood.